The activities of three enzymes and the concentration of intermediates involved in the synthesis of N,N-dimethyltryptamine (DMT) from endogenous tryptophan (TRP) have been measured in vitro in seedlings of Phalaris aquaticaL. cv Australian Commercial over 16 days after planting. The activities of tryptophandecarboxylase and the two N-methyltransferases increased rapidly to maximal rates of substrate conversion at day 5 of 95, 1000, and 2200 micromoles per hour per milliliter, respectively. After these maximal rates, the activities decreased rapidly. The concentration of intermediates increased rapidly from zero in the seeds to maximalvalues of 25 and 53 micromolar at day 5 for tryptamine (T) and N-methyltryptamine (MT), respectively, 1000 micromolar at day 6 for TRP, and 650 micromolar at day 8 for DMT. The concentration of DMT and of all the intermediates in its synthesis declined rapidly after the maximal value had been reached. A mathematical model of the pathway from TRP to DMT using these enzymes correctly predicts the concentrations of T and MT, intermediates whose concentration is determined only by the pathway, and confirms that these three enzymes are responsible for the in vivo synthesis of DMT. Kinetic studies are reported for these enzymes.